Julang Li

In vitro germline potential of stem cells derived from fetal porcine skin

Two of the unanswered questions in mammalian developmental biology are when and where the fate of the germ cell is specified. Here, we report that stem cells isolated from the skin of porcine fetuses have the intrinsic ability to differentiate into oocyte-like cells. When differentiation was induced, a subpopulation of these cells expressed markers such as Oct4, Growth differentiation factor 9b (GDF9b), the Deleted in Azoospermia-like (DAZL) gene and Vasa — all consistent with germ-cell formation. On further differentiation, these cells formed follicle-like aggregates that secreted oestradiol and progesterone and responded to gonadotropin stimulation. Some of these aggregates extruded large oocyte-like cells that expressed oocyte markers, such as zona pellucida, and the meiosis marker, synaptonemal complex protein 3 (SCP3). Some of these oocyte-like cells spontaneously developed into parthenogenetic embryo-like structures. The ability to generate oocyte-like cells from skin-derived cells may offer new possibilities for tissue therapy and provide a new in vitro model to study germ-cell formation and oogenesis.

Leptin enhances porcine preimplantation embryo development in vitro

Recent studies have suggested that leptin plays an important role in reproduction. Ob-R is expressed in the murine embryo, and is suggested to play a role in embryo development, although contradictory results have been reported. In the present study, Ob-R expression was observed both at the mRNA and protein levels in porcine early embryos. We have also demonstrated that leptin is produced in the porcine oviduct, making it spatially available to interact with its receptor during preimplantation development. When included at 10 ng/ml in embryo culture medium, leptin significantly increased the proportion of cleaved embryos (P < 0.01). At day 7 of in vitro culture, leptin at 10 and 100 ng/ml increased the proportion of embryos reaching the blastocyst stage (P < 0.01). We have previously observed that leptin increases oocyte maturation in vitro, and here we report that inclusion of leptin in both IVM and embryo culture medium further increased blastocyst development (P < 0.05), compared to when leptin was included in the embryo culture alone, suggesting leptin has a synergistic role on both oocyte maturation and preimplantation embryo development.

Primordial germ cell-like cells differentiated in vitro from skin-derived stem cells.

Background We have previously demonstrated that stem cells isolated from fetal porcine skin have the potential to form oocyte-like cells (OLCs) in vitro. However, primordial germ cells (PGCs), which must also be specified during the stem cell differentiation to give rise to these putative oocytes at more advanced stages of culture, were not systematically characterized. The current study tested the hypothesis that a morphologically distinct population of cells derived from skin stem cells prior to OLC formation corresponds to putative PGCs, which differentiate further into more mature gametes. Methodology/Principal Findings When induced to differentiate in an appropriate microenvironment, a subpopulation of morphologically distinct cells, some of which are alkaline phosphatase (AP)-positive, also express Oct4, Fragilis, Stella, Dazl, and Vasa, which are markers indicative of germ cell formation. A known differentially methylated region (DMR) within the H19 gene locus, which is demethylated in oocytes after establishment of the maternal imprint, is hypomethylated in PGC-like cells compared to undifferentiated skin-derived stem cells, suggesting that the putative germ cell population undergoes imprint erasure. Additional evidence supporting the germ cell identity of in vitro-generated PGC-like cells is that, when labeled with a Dazl-GFP reporter, these cells further differentiate into GFP-positive OLCs. Significance The ability to generate germ cell precursors from somatic stem cells may provide an in vitro model to study some of the unanswered questions surrounding early germ cell formation.

In Vitro and In Vivo Germ Line Potential of Stem Cells Derived from Newborn Mouse Skin

We previously reported that fetal porcine skin-derived stem cells were capable of differentiation into oocyte-like cells (OLCs). Here we report that newborn mice skin-derived stem cells are also capable of differentiating into early OLCs. Using stem cells from mice that are transgenic for Oct4 germline distal enhancer-GFP, germ cells resulting from their differentiation are expected to be GFP+. After differentiation, some GFP+ OLCs reached 40–45 µM and expressed oocyte markers. Flow cytometric analysis revealed that ∼0.3% of the freshly isolated skin cells were GFP+. The GFP-positive cells increased to ∼7% after differentiation, suggesting that the GFP+ cells could be of in vivo origin, but are more likely induced upon being cultured in vitro. To study the in vivo germ cell potential of skin-derived cells, they were aggregated with newborn ovarian cells, and transplanted under the kidney capsule of ovariectomized mice. GFP+ oocytes were identified within a subpopulation of follicles in the resulting growth. Our finding that early oocytes can be differentiated from mice skin-derived cells in defined medium may offer a new in vitro model to study germ cell formation and oogenesis.

Embryos Derived from Porcine Skin-Derived Stem Cells Exhibit Enhanced Preimplantation Development

Abstract Ongoing research to identify the most suitable type of donor cell for nuclear transfer (NT) has suggested that less differentiated stem cells may be better donors than other somatic cell types. Recently, we have reported the isolation and characterization of porcine skin-originated sphere (PSOS) stem cells from fetal skin, making it possible to test this hypothesis in a nonrodent animal model. In the present study, we have investigated and compared the feasibility and preimplantation developmental efficiency of using fetal PSOS cells and fibroblasts as nuclear-transfer donors. The majority of fetal PSOS cells are in the G1/ G0 stage of the cell cycle, which is desirable for NT. During long-term in vitro culture, fetal PSOS cells had greater genome stability, with a lower frequency of abnormal karyotypes than fetal fibroblast cells. Embryos cloned from PSOS cells showed enhanced preimplantation development compared with fibroblast cloned embryos, which is indicated by an increased rate of blastocyst development and a higher total cell number in Day 7 blastocysts. The gene expression profile of genes critical for early development from eight-cell-stage PSOS NT embryos more closely resembled the pattern observed from in vivo-produced embryos compared with that of fibroblast-cloned embryos. Cumulatively, our data suggest that fetal PSOS cells may be better donor cells for NT in the pig.

Epidermal Growth Factor-Expressing Lactococcus lactis Enhances Intestinal Development of Early-Weaned Pigs

Stress and incomplete gastrointestinal development in early-weaned piglets represent significant challenges in commercial swine farming. Orally ingested recombinant epidermal growth factor (EGF) has been shown to remain biologically active in the gastrointestinal tract as well as stimulate intestinal development, reducing the incidence of pathogen infection and diarrhea. We have previously shown that the food-grade bacterium Lactococcus lactis can be genetically altered to express biologically active EGF when fed to early-weaned mice. In this study, we assigned 8 pigs to each of 4 groups that were given EGF-expressing L. lactis (EGF-LL), empty vector-expressing L. lactis (EV-LL), recombinant human EGF, or unsupplemented bacterial media, all of which were delivered as 50-mL i.g. doses twice per day. All pigs were killed after 14 d to examine intestinal morphology. Pigs in the EGF-LL group had greater jejunal and duodenal villus heights (P < 0.0001) and intestinal length (P = 0.049) than pigs in the control group. Immunohistochemistry with antibodies against proliferating cell nuclear antigen (PCNA) revealed that the proliferation of intestinal cells was significantly greater in the EGF-LL group than in the control group. PCNA expression and intestinal length also were greater in the EV-LL group, which received L. lactis that did not express EGF, than in the control group (P = 0.049), further supporting the use of naturally occurring intestinal microbes as desirable vectors for recombinant protein delivery. Our data demonstrates the feasibility of delivering a growth factor using common probiotic bacteria to farm animals for commercial practice.

A growth-maturation system that enhances the meiotic and developmental competence of porcine oocytes isolated from small follicles.

Abstract In livestock, most of the follicles on the ovarian surface are small follicles. A procedure that supports the in vitro growth and maturation of these small follicle-derived oocytes may offer a new source of useable oocytes for both biotechnological and fundamental research purposes. The objective of the current study was to test the hypothesis that providing a more growth-supporting and less maturation-promoting environment during the first phase of small follicle-derived oocyte maturation may improve oocyte competence for meiosis and embryo development upon activation. In our small follicle-derived oocyte growth-maturation system (SGM group), cumulus-oocyte complexes (COCs) from small follicles (1–3 mm) were first cultured in oocyte growth medium for 24 h, then in oocyte maturation medium for 20 h. As controls, COCs from small (SM group) and large (LM group) follicles were cultured using a conventional in vitro maturation (IVM) approach in which they were directly cultured in oocyte maturation medium. At 24 h of culture, the percentage of small follicle-derived oocytes that underwent germinal vesicle breakdown (GVBD) in the SGM group was comparable to that of large follicle-derived oocytes (LM group) but was significantly higher than that of the SM group (P < 0.05). At 44 h of culture, compared to 36% in the SM group, 55% of the SGM group oocytes reached metaphase II (MII; P < 0.05). In addition, the level of cyclin B in oocytes of the SGM group was comparable to that of oocytes from LM group and was significantly higher than that of oocytes from the SM group (P < 0.05). When activated and in vitro fertilized (IVF), 7.3 and 9.0 times more parthenogenetic and IVF embryos developed to blastocyst stage in the SGM group than in the SM group (P < 0.05). The mRNA expression levels of three developmentally important genes—DNA-methyltransferase 1, Pou domain class 5 transcription factor 1, and Fibroblast growth factor receptor 2—in embryos of the SGM group were comparable to those of embryos developed from the LM group, whereas they were significantly lower in those of the SM group (P < 0.05). Our data suggest that the oocyte growth-maturation system facilitates the final stage of oocyte growth and thus resulted in better oocyte nuclear, cytoplasmic maturation, and developmental competency compared with the conventional direct oocyte maturation system.

Adverse effects induced by short hairpin RNA expression in porcine fetal fibroblasts

RNA interference is a recent, gene silencing technique that could be extremely valuable in studying gene function, treating diseases, and developing novel animal models for human diseases. Here, we investigated the feasibility of applying shRNA-mediated RNA interference in fetal fibroblasts for silencing of the myostatin gene and investigate adverse effects of RNAi. We report that up to 97% silencing of myostatin mRNA was achieved using shRNA constructs in transiently and stably transfected fetal fibroblasts (p<0.05). At the same time we also demonstrate that high level of shRNA expression resulted in 10- to 1000-fold induction of interferon responsive genes (OAS1, IFN-beta) (p<0.05). In addition we also report novel adverse effect of shRNA expression in stably transfected cells-interference with microRNA processing/transport which led to 500-fold increase in the level of miR21 precursors (p<0.05). Reduction of these side effects will be essential to obtain long term stable RNAi silencing.

Analysis of Oocyte-Like Cells Differentiated from Porcine Fetal Skin-Derived Stem Cells

We previously reported the differentiation of cells derived from porcine female fetal skin into cells resembling germ cells and oocytes. A subpopulation of these cells expressed germ cell markers and formed aggregates resembling cumulus-oocyte complexes. Some of these aggregates extruded large oocyte-like cells (OLCs) that expressed markers consistent with those of oocytes. The objective of the current study was to further characterize OLCs differentiated from porcine skin-derived stem cells. Reverse transcriptase (RT)-polymerase chain reaction and Western blot revealed the expression of connexin37 and connexin43, both of which are characteristic of ovarian follicles. The expression of meiosis markers DMC1 and synaptonemal complex protein, but not STRA8 and REC8, was detected in the OLC cultures. Immunofluorescence with an antibody against synaptonemal complex protein on chromosome spreads revealed a very small subpopulation of stained OLCs that had a similar pattern to leptotene, zytotene, or pachytene nuclei during prophase I of meiosis. Sodium bisulfite sequencing of the differentially methylated region of H19 indicated that this region is almost completely demethylated in OLCs, similar to in vivo-derived oocytes. We also investigated the differentiation potential of male skin-derived stem cells in the same differentiation medium. Large cells with oocyte morphology were generated in the male stem cell differentiation cultures. These OLCs expressed oocyte genes such as octamer-binding transcription factor 4 (OCT4), growth differentiation factor-9b (GDF9B), deleted in azoospermia-like (DAZL), VASA, zona pellucida B (ZPB), and zona pellucida C (ZPC). It was concluded that skin-derived stem cells from both male and female porcine fetuses are capable of entering an oocyte differentiation pathway, but the culture system currently in place is inadequate to support the complete development of competent oocytes.

MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells

We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion (HAS2, PTGS2) and oocyte maturation (CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zona pellucida 3 (ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation.